Dr. Aadel Chaudhuri is an Assistant Professor of Radiation Oncology at Washington University. The NCI K08 award will provide the funding necessary to achieve important goals for his career development, by allowing him to: 1) Demonstrate that circulating tumor DNA can be used as an MRD biomarker after definitive-intent treatment of localized liver cancer, 2) To use circulating tumor DNA as an early response biomarker for liver cancer immune checkpoint blockade, and 3) To infer mechanisms of immunotherapy response by deconvolution of cell-free RNA confirmed by single cell sequencing. To oversee Dr. Chaudhuri's training, he will be mentored by Dr. Dennis Hallahan, an established figure in cancer biology and radiation oncology, Dr. Maximilian Diehn, an expert in cell-free nucleic acid translational research, and by Dr. Timothy Ley, a world- renowned expert in cancer genomics. In addition to his mentors, Dr. Chaudhuri has assembled a group of advisors/collaborators who will lend expertise to his proposed research on cell-free nucleic acids for response assessment of hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is the number three cause of cancer death worldwide and stereotactic body radiotherapy (SBRT) and interventional radiology (IR) ablation play major roles in the definitive management of early stages of disease. This proposal aims to address major clinical challenges for HCC patients treated with SBRT or IR ablation. First, there is no standard-of-care surveillance modality that can reliably detect molecular residual disease (MRD) after completion of therapy. Second, loco-regional post-treatment inflammatory/fibrotic tissue changes can often be difficult to distinguish from tumor recurrence on standard-of-care surveillance imaging. Thirdly, there is no standard-of-care modality available for assessing immune checkpoint blockade early. To address these clinical challenges, we plan to use a method for profiling cell-free DNA that my mentor's laboratory developed called CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), which relies on capture-based next-generation sequencing, and an analogous method for profiling cell- free RNA. CAPP-Seq allows ultrasensitive quantitation of circulating tumor DNA (ctDNA) and through tracking of multiple mutations and integrated digital error suppression has a detection limit of ~10 parts per million. We recently published that CAPP-Seq ctDNA analysis can reliably detect molecular residual disease (MRD) shortly after localized lung cancer treatment completion, and can be used for immune checkpoint inhibitor early response assessment for metastatic lung cancer (MS in preparation). We hypothesize that similar methods can be applied to localized HCC to detect post-treatment MRD, and for response assessment and mechanism elucidation for advanced HCC patients treated with immune checkpoint blockade.